Fungi are disease-causing agents in plants and affect crops of economic importance. One control method is to induce resistance in the host by using biological control with hypovirulent phytopathogenic fungi. Here, we report the detection of a mycovirus in a strain of Colletotrichum gloeosporioides causing anthracnose of cashew tree. The strain C. gloeosporioides URM 4903 was isolated from a cashew tree (Anacardium occidentale) in Igarassu, PE, Brazil. After nucleic acid extraction and electrophoresis, the band corresponding to a possible double-stranded RNA (dsRNA) was purified by cellulose column chromatography. Nine extrachromosomal bands were obtained. Enzymatic digestion with DNAse I and Nuclease S1 had no effect on these bands, indicating their dsRNA nature. Transmission electron microscopic examination of extracts from this strain showed the presence of isometric particles (30-35 nm in diameter). These data strongly suggest the infection of this C. gloeosporioides strain by a dsRNA mycovirus. Once the hypovirulence of this strain is confirmed, the strain may be used for the biological control of cashew anthracnose.

Fungi are the most important disease-causing agents in plants with serious economic impact. Chemical methods to control phytopathogenic fungi can affect human health and have environmental consequences. In an attempt to minimize dependence on chemical methods, alternative means of pathogen control have been investigated (Yu et al., 2010). One alternative is to use hypovirulent phytopathogenic fungal strains that can biologically control phytopathogenic fungi of the same or related species. Fungal hypovirulence is usually caused by mycoviral infection. Hypovirulent fungi cause milder infection and may induce hypovirulence in other pathogenic strains via viral transfer (Pearson et al., 2009).

All currently known mycoviruses have isometric, double-stranded RNA (dsRNA)-containing particles, and are classified into three families: Chrysoviridae, Endornaviridae, Partitiviridae and Totiviridae. Particle size ranges from 25 to 50 nm in diameter, except for endornaviruses which do not produce virions (Chu et al., 2002; Ghabrial & Suzuki, 2009).

In this communication, we report the presence of isometric dsRNA viral particles in a strain of Colletotrichum gloeosporioides (Penz.) Sacc., the causal agent of anthracnose in cashew tree.

Colletotrichum gloeosporioides URM 4903 was isolated from anthracnose lesions on a cashew tree (Anacardium occidentale L.) in the town of Igarassu, Pernambuco, Brazil (7°50'3.74"S and 34°54'22.87"O), and deposited in the mycology collection (URM) at Federal University of Pernambuco, Recife, PE. This strain was chosen after preliminary tests revealed poor pathogenicity in detached cashew leaves and the presence of extrachromosomal bands in agarose gel electrophoresis (unpublished data).

PDA (20% potato extract, 2% dextrose, 1.5% agar) and PD (20% potato extract, 2% dextrose) media were used to grow this C. gloeosporioides strain. The strain, when needed, was incubated at 28°C for 7 days before use.

Mycelia for nucleic acid extraction were obtained after inoculating PD medium with conidia of the C. gloeosporioides strain. After 3 days of continuous shaking, mycelia were filtered and ground to a fine powder with liquid nitrogen. Total nucleic acids were extracted as described by Raeder & Broda (1985) and analyzed by agarose gel electrophoresis at 3 Vcm-1 and ethidium bromide staining (0.5 µg mL-1).

Bands corresponding to the total fungal nucleic acids were obtained after agarose gel electrophoresis (Figure 1A). They were purified by cellulose column chromatography according to the method of Morris & Dodds (1979) with modifications. The cellulose column was mounted on a disposable 10 mL syringe, with 2.5 g of microgranular cellulose evenly distributed. The column was buffered with successive washings with STE buffer (0.1 M NaCl, 0.05 M Tris, 0.001 M NaEDTA, pH 7.0) and 15% ethanol. After sample application, the column was washed with STE-15% ethanol to remove DNA and single-stranded RNA. The putative dsRNA that remained in the column was eluted with STE without ethanol and precipitated by addition of 0.1 volume of 3 M sodium acetate and 2 volumes of absolute ethanol (-20°C) for approximately 12 h. The precipitate was collected by centrifugation at 10,000 g for 15 min. The supernatant was discarded, and the precipitate was washed with 70% ethanol, dried in a laminar flow cabinet and suspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The several stages of chromatography were visualized after agarose gel electrophoresis.

After chromatography, the purified material was digested with DNAse I and nuclease S1. Nuclease S1 (35 U/mg) digestion was performed at 37°C for 30 min. DNAse I (20 mg/mL in 30 mM MgCl2) digestion was performed at 37°C for 20 min, followed by incubation at 65°C for 15 min to inactivate the enzyme. Digestion results were analyzed by 1% agarose gel electrophoresis as previously described.

We attempted to visualize the putative mycovirus in the C. gloeosporioides strain by examining negatively stained mycelial extracts by transmission electron microscopy. Hyphae and spores of the fungus grown in PDA medium were carefully removed with a sterile needle, transferred to microtubes containing small spherical glass beads, and homogenized in 0.2 mL 0.05 M cacodylate buffer (pH 7.2). A small drop of this material was placed on a parafilm sheet, and Formvar/carbon-coated 300 mesh grids were floated on it for 15 min. The grids were removed, and excess liquid was removed with a piece of filter paper. The grids were washed with drops of distilled water and floated on a droplet of 1% aqueous uranyl acetate for 5 min. After removal of excess liquid, the grids were dried and examined in a Zeiss EM 900 transmission electron microscope with digital image recording. Although in low concentrations, isometric particles (30-35 nm in diameter) were consistently found in the fungal extracts (Figure 1B). They were essentially similar to other mycoviruses in the Chrysoviridae, Partitiviridae and Totiviridae families, whose diameters range from 25 to 50 nm and the genomes are comprised of dsRNA molecules.

The presence of dsRNA in fungi can be demonstrated by several processes after total nucleic acid extraction and purification. The C. gloeosporioides strain isolated from cashew tree showed extrachromosomal bands after total nucleic acid electrophoresis (Figure 1A). After cellulose column chromatography we observed nine bands, all of which were resistant to DNAse I and Nuclease SI digestion, indicating that they consisted of dsRNA (Figure 1C).

To the best of our knowledge, dsRNA has been reported only once in Colletotrichum lindemuthianum (Rawlinston et al., 1975) and C. gloeosporioides, which cause different anthracnose diseases on Stylosanthes (Dale et al., 1988). This study is the first to report a possible dsRNA virus in C. gloeosporioides causing anthracnose of cashew tree. Once the hypovirulence of C. gloeosporioides URM 4903 has been proven, we may test its utility as an alternative biological control agent for cashew tree anthracnose.

ACKNOWLEDGEMENTS

We thank the staff of the Microorganism Genetics Laboratory of the Federal University of Amazonas (UFAM), who allowed us to use their facilities and instruments. We also thank the Brazilian Research Council (CNPq) for supporting grants to Lívio C. de Figueirêdo and the Postgraduate Program in Biotechnology of UFAM for supporting this project.